The amino acid L-glutamate elicits a unique taste sensation in humans called umami (delicious taste) by Asian cultures. An important feature of the umami sensation is that it is potentiated several fold by the presence 5'-ribonucleotides. The receptors involved in glutamate taste and the mechanisms involved in their modulation by 5'-ribonucleotides are not known. Using a combination of patch-clamp recording from isolated rat taste buds and recording from glutamate receptors expressed in Xenopus oocytes and heterologous mammalian cell lines, we will test the following hypothesis: Specific membrane receptors for glutamate are found within taste bud cells and serve to transduce the taste of glutamate. The specific aims are as follows: (1) To determine the properties of endogenous glutamate receptors in taste cells. What are the kinetic and pharmacological properties of glutamate- activated currents in taste cells? Are glutamate-activated currents mimicked by particular glutamate agonists? Are glutamate-activated currents potentiated by 5'-ribonucleotides? What are the single-channel properties of glutamate receptors or their ion channel targets in taste cells? Although we will initially focus on metabotropic receptors, we will characterize all glutamate-activated currents observed in taste cells. Whole-cell and single-channel recording techniques will be utilized. (2) To determine the location of endogenous glutamate receptors in taste cells. Are glutamate receptors of a particular subtype located preferentially in apical? Glutamate and various agonists, together with 5'-ribonucleotides will be applied selectively to apical or basolateral membranes using a special mounted preparation for whole cell recording. (3) To determine the functional properties of glutamate receptors encoded by taste cell cDNAs. Do the cloned glutamate receptors have physiological and pharmacological properties similar to the endogenous glutamate receptors found in taste cells? Glutamate receptor cDNAs will be transfected into heterologous mammalian cell lines, and in vitro- transcribed mRNAs will be injected into Xenopus oocytes. Properties of the expressed receptors will be assessed by patch-clamp recording, cAMP radioimmunoassay, and two-electrode voltage-clamp. (Collaboration with Project #1, Chaudhari) These studies will provide important new information about the functional significance of glutamate receptors in taste buds, including the types of glutamate receptors involved, their location on taste receptor cells, and the mechanisms involved in their modulation by 5'-ribonucleotides.